N6-methyladenosine (m6A) was recently discovered to be a widespread base modification present in thousands of mammalian mRNAs. In addition to its prevalence throughout the transcriptome, researchers have also revealed that m6A is a reversible modification, suggesting that dynamic regulation of mRNA methylation is a novel and widespread RNA regulatory mechanism in cells. Much of our understanding of the physiological importance of m6A comes from studies of m6A demethylases. Indeed, disruption of the m6A demethylase FTO leads to dysfunction of dopaminergic pathways in the brain and an abnormal response to cocaine in mice. Additionally, FTO genetic mutations have been strongly associated with diseases including melanoma and breast cancer in humans. Despite the importance of FTO for human health and disease, tools which directly measure FTO activity have not been developed. In this proposal, we use a simple yet innovative RNA aptamer-based strategy to develop assays which measure the activity of FTO and other m6A demethylating enzymes. The results of this Phase I SBIR will be the development of an optimized fluorescence assay for measuring m6A demethylase activity and demonstration of the compatibility of this tool for high-throughput screening of small-molecule FTO inhibitors. This application will enable us to develop the first commercially-viable assay for measuring FTO activity, which will fulfill an important need in the RNA methylation and FTO research communities. Furthermore, these experiments will enable the development of HTS assays for the identification of novel inhibitors of FTO and other RNA demethylase enzymes.